Pirazolone 3-Carboxylic Acid PDF

Article
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Identication of High-Anity P2Y12 Antagonists Based on a

Phenylpyrazole Glutamic Acid Piperazine Backbone
Gernot Zech, Gerhard Hessler, Andreas Evers, Tilo Weiss, Peter Florian, Melitta Just, Jorg Czech,
Werngard Czechtizky, Jochen Gorlitzer, Sven Ruf, Markus Kohlmann, and Marc Nazare*
Sano-Aventis Deutschland GmbH, Industriepark Hochst, Building G878, D-65926 Frankfurt am Main, Germany
S Supporting Information
*
ABSTRACT: A series of novel, highly potent P2Y12 antagonists as

inhibitors of platelet aggregation based on a phenylpyrazole glutamic
acid piperazine backbone is described. Exploration of the structural
requirements of the substituents by probing the structureactivity
relationship along this backbone led to the discovery of the N-acetyl(S)-proline cyclobutyl amide moiety as a highly privileged motif.
Combining the most favorable substituents led to remarkably potent
P2Y12 antagonists displaying not only low nanomolar binding anity
to the P2Y12 receptor but also a low nanomolar inhibition of platelet
aggregation in the human platelet rich plasma assay with IC50 values
below 50 nM. Using a homology and a three-dimensional
quantitative structureactivity relationship model, a binding
hypothesis elucidating the impact of several structural features was
developed.
1. INTRODUCTION
The widespread successful clinical use of the irreversible

P2Y12 antagonist clopidogrel5 has demonstrated the high
impact and relevance of inhibiting this mechanism for the
prevention of thrombotic complications of atherosclerosis.
Albeit being highly selective, clopidogrel requires hepatic
metabolic conversion to an active metabolite that covalently
modies the P2Y12 receptor, which results in a slow onset and
oset of its pharmacological action.6 Consequently, extensive
drug discovery eorts were made to identify potent, directacting, and reversible P2Y12 antagonists resulting in promising
compounds like Ticagrelor (AZD-6140) (1),7 Elinogrel (PRT128) (2),8 and BX 667 (3)9 (Chart 1).
The activity data for the majority of reversible antagonists
show that, despite high binding anity to the P2Y12 receptor,
the inhibitory activity in the clinical relevant functional human
platelet-rich plasma (hPRP) assay, which is a key predictor for
the antithrombotic pharmacological eect,10 still leaves
signicant room for improvement. For example, when
evaluating the antiaggregatory activity in hPRP of 13 in our
standard assay system, which was used throughout this
investigation, potencies in the micromolar range were
determined. However, improving the P2Y12-mediated inhibitory potency in the hPRP assay setting remains a signicant
challenge as the observed activity is governed by the complex
interplay of several parameters such as receptor binding anity,
plasma protein binding, and polarity of the antagonist.11
The activation and aggregation of blood platelets are essential

for normal hemostasis but are also key mechanisms for
triggering acute, potentially life-threatening arterial thrombotic
events like myocardial infarction or thromboembolic stroke. In
this context, adenosine-5-diphosphate (ADP) is a central
signaling molecule inducing platelet activation and aggregation,
thus playing a key role in the initiation and progression of
arterial thrombus formation. Upon activation by various stimuli,
like collagen (Col) and thrombin (Thr), ADP is released from
blood platelets in the vasculature, as well as from damaged
blood cells, endothelium, or tissues.1 The ADP-induced platelet
aggregation is triggered by its binding to two specic G-proteincoupled receptors (GPCRs), P2Y purinoceptor 1 (P2Y1) and
P2Y purinoceptor 12 (P2Y12), which are expressed on the
plasma membrane of human platelets. ADP binding to these
receptors induces inhibition of adenylyl cyclase and modulation
of intracellular signaling pathways such as inux and
mobilization of intracellular Ca2+, activation of phosphoinositide-3 kinase (PI3K), shape change, platelet aggregation, and
secretion of other mediators, thus amplifying the initial
proaggregatory signal.2 In particular the P2Y12 receptor has
been recognized as a highly attractive target, as its activation is
required for platelet secretion and stabilization of platelet
aggregates, and in contrast to the ubiquitously expressed P2Y1
receptor, the P2Y12 receptor is mainly expressed on platelets.3
Moreover, experimental studies have shown that sole inhibition
of the P2Y12 receptor is sucient to block and prevent platelet
aggregation.4
2012 American Chemical Society
Received: June 4, 2012

Published: September 17, 2012
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dx.doi.org/10.1021/jm300771j | J. Med. Chem. 2012, 55, 86158629
Journal of Medicinal Chemistry
Article
Chart 1. Structures of 1 (Ticagrelor), 2 (Elinogrel), and 3 (BX 667) and Their Respective Anity in the P2Y12 Binding Assay
and Antiaggregatory Activity in the Human Platelet-Rich Plasma (hPRP) Assay Used Throughout This Investigation (See the
Experimental Section)
Figure 1. (a) Binding hypothesis of quinoline 3 in the P2Y12 receptor obtained from docking into a receptor homology model. (b) Threedimensional alignment of quinoline 3 and phenylpyrazolone 4.
In seeking suitable P2Y12 drug candidates in this area, we12

along with others9,11,13 have explored glutamic acid piperazine
derivatives as reversible P2Y12 antagonists. Here, we report our
ndings on the identication of novel, highly potent P2Y12
antagonists based on a phenylpyrazolone scaold and the
discovery of new high-anity ligand substitution patterns
resulting in a dramatic enhancement in binding anity and
functional activity.
receptor type 4 (CXCR4) receptor as a structural template (see

the Experimental Section), which had previously been
identied as the most-suited structural template.14 Two main
factors limit the accuracy of the P2Y12 protein model, namely,
the relatively low sequence identity of 25.6% in the transmembrane region and the diverse orientations of the
extracellular 2 (EC2) loop linked to transmembrane helix 3
(TM3) by a cysteine bridge. On the basis of the analysis of
dierent GPCR crystal structures, it has been shown that
residues from this loop region participate in ligand binding in
dierent GPCRs.15 Consequently, we had to take into account
that the EC2 loop orientation is dierent from that observed in
the CXCR4 template. To compensate for these factors, limiting
the accuracy of the protein model, we cross-checked the
docking mode with available mutagenesis and ligand SAR data.
Furthermore, we used the docking modes of quinoline lead 3
and phenylpyrazolone 4 as the structural basis for the
generation of multiple ligand alignments and derivation of
ligand-based 3D quantitative structureactivity relationship
(QSAR) models.
Figure 1a shows the binding hypothesis for 3, which was
obtained from molecular docking into our homology model of
the P2Y12 receptor. The quinoline lead 3 binds into a cavity
formed by residues from TM3, TM4, TM5, TM6, and TM7.
The carboxy group is coordinated by the basic side chains of
residues Arg6.55 and Lys7.35. Whereas the docking mode
proposes a localization of the 4-hydroxy-quinoline moiety in a
hydrophobic subpocket, the piperazinyl carbamate substituent
2. RESULTS AND DISCUSSION

2.1. Identication of the Phenylpyrazolone Scaold
by Structure-Based Alignment Studies. As a result of
initial alignment considerations in the context of P2Y12
antagonists of the glutamic acid piperazine type, we anticipated
that a phenylpyrazolone scaold12 as shown in compound 4
could be a suitable replacement for the initial quinoline scaold
in compound 3 (Figure 1). To further rationalize our ligand
design eorts and direct our structureactivity relationship
(SAR) investigation, we intended to use structure-based
alignments. However, although tremendous progress has been
made in revealing 3D structures of GPCRs, the investigation of
3D structures of membrane proteins remains a signicant
challenge, and there is currently no P2Y12 X-ray crystal
structure available. Therefore, we resorted to the use of
homology modeling to gain structural insights into protein
ligand interactions for P2Y12. For the generation of binding
hypotheses, a homology model of the P2Y12 receptor was
created using the crystal structure of the C-X-C chemokine
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Figure 2. (a) Schematic representation of the regions to be investigated based on the phenylpyrazolone backbone. (b) Proposed binding mode of
pyrazole 4 into the P2Y12 homology model.
Scheme 1. Synthetic Routes to Key Building Blocksa
points toward the extracellular region. Interestingly, a similar

docking hypothesis was recently described for a related
chemical series of piperazinyl glutamic acid pyrimidines.16
Pyrazole 4 was docked in to the P2Y12 homology model as well
(Figure 1b). As shown, the large identical glutamic acid
piperazinyl carbamate fragments align well. The phenylpyrazolone moiety is placed in the same hydrophobic pocket
as the quinoline fragment. However, a more careful inspection
of the 3D alignment of 3 with 4 reveals that the substituent at
the 5-position of the pyrazole 4 does not align with the
corresponding substituent at the 4-position of the quinoline 3
(see Figure 1b). Thus, the substantial topological change from
the quinoline to the pyrazole scaold obviously also required an
examination of the remaining regions of the ligand.
Figure 2a gives a schematic representation of the regions that
we intended to explore based on the phenylpyrazole glutamic
acid piperazine backbone. Analysis of the docking mode of
pyrazole 4 (Figure 2b) suggested that in particular the 5position of the pyrazole should be a promising region for
further variations. Consequently, we rst set out to systematically probe the inuence of variations at the 5-position of the
phenylpyrazole. The glutamic acid side chain was kept constant
throughout these initial variations serving as a strong mediator
of anity for the putative antagonists.
Reagents and conditions: (a) TOTU, N-ethylmorpholine, DMF,

room temp, 16 h, 90%. (b) H2 (3 bar), Pd/C (10%), ethanol, room
temp, 6 h, 90%. (c) Benzyl 2-bromoacetate, Cs2CO3, DMF, room
temp, 12 h, 95%. (d) H2 (3 bar), Pd/C (10%), EtOAc, room temp, 6
h, 76%. (e) Benzyl (2S)-pyrrolidine-2-carboxylate, EDC, HOBt,
EtNiPr2, DMF, room temp, 16 h, 84%. (f) H2 (3 bar), Pd/C (10%),
EtOAc, room temp, 6 h, 98%. (g) Cyclobutyl amine, HATU, EtNiPr2,
DMF, room temp, 16 h, 95%. (h) NaOH, H2O/THF, room temp, 16
h, 95%.
3. CHEMISTRY
Scheme 1 shows the synthesis of representative building blocks,
which were sequentially attached to the 3-carboxyphenylpyrazole, enabling a highly convergent and exible synthesis of the
nal ligands. The glutamic piperazinyl carbamate building block
7 was prepared from the commercially available Z-O-tert-butyl
protected 5 in two steps by standard amide coupling using O((ethoxycarbonyl)cyanomethyleneamino)-N,N,N,N-tetramethyluronium tetrauoroborate (TOTU) followed by the
hydrogenolytic removal of the CbZ-protecting group (sequence
a). The second key intermediate used throughout this
investigation was the fully decorated phenylpyrazole building
block 10 (sequence b). This fragment was synthesized by
subjecting the phenylpyrazole ester 8 to an O-alkylation with
benzyl 2-bromoacetate followed by selective hydrogenolytic
cleavage of the benzylester to give 9. This intermediate was
then converted to the acid 10 by a high-yielding sequence of
amide coupling and deprotection steps.
The syntheses of the fully decorated nal phenylpyrazolone
ligands with dierent substituents attached to the 5-position of
the pyrazole and all other side chain variations were carried out
by two optional routes starting from commercially available 3carboxy phenyl pyrazolones 8 and 11 (Scheme 2).
Phenylpyrazolone variations keeping the piperazinyl side
chain constant were synthesized according to route 1.
Phenylpyrazolones of type 14 were prepared by using standard
amide coupling conditions with 1-ethyl-3-(3dimethylaminopropyl)carbodiimide hydrochloride (EDC)/1hydroxybenzotriazole (HOBt) followed by O-alkylation of the
phenylpyrazole with benzyl 2-bromoacetate in the presence of
cesium carbonate in DMF and hydrogenolytic deprotection to
the carboxylic acid 12. The diversifying amide variation step
was then conducted using EDC/HOBt or O-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexauorophosphate
(HATU) in DMF as activating reagents.
Route 2 was used to explore the impact of other side chain
variations retaining the 5-O-alkyl amide substituent. The
preassembled phenylpyrazole 15 and the piperazinyl side
chain 16 were prepared according to the synthetic sequences
outlined for the respective building blocks (Scheme 1, synthesis
of 7 and 10) and then coupled using either EDC/HOBt or
HATU activation in DMF to give the nal derivatives 14. By
using these synthetic routes, we were able to systematically
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Scheme 2. Synthetic Routes Used for the Assembly of the

Final P2Y12 Antagonistsa
Table 1. Binding and hPRP Activity of Explorative

Variations around the Initial Hit 4
Reagents and conditions: (a) EDC, HOBt, EtNiPr2, 16, DMF, room
temp. (b) Benzyl 2-bromoacetate, Cs2CO3, DMF, room temp. (c) H2
(3 bar), Pd/C (10%), EtOAc, room temp. (d) EDC, HOBt, EtNiPr2,
DMF, room temp or HATU, EtNiPr2, DMF, room temp. (e) NaOH,
H2O/THF, room temp.
synthesize a wide range of P2Y12 antagonists and to obtain a

dense set of SAR data, which revealed key features of favorable
and unfavorable interactions.
3. RESULTS AND DISCUSSION

The binding anity of the compounds was determined by a
human P2Y12 receptor binding assay employing [33P]2-MeSADP as the radioligand and human P2Y12 transfected Chinese
hamster ovary (CHO) cell membranes.17 The inhibitory eect
on the ADP-stimulated platelet aggregation was determined in
vitro in hPRP using a turbidimetric method in a 96-well format.
For several compounds, the antiaggregatory activity was
additionally conrmed as indicated in a second hPRP assay,
the clinically relevant Born method using single cuvettes.10
Table 1 shows the SAR of an initial explorative library intended
to examine the eects of various ester, ketone, and amide
derivatizations obtained by route 1 transformations.
This survey illustrated the fact that the receptor recognition
was highly sensitive to modications at the 5-position of the
pyrazole, whichaccording to our docking hypothesispoints
into a buried subpocket composed of several hydrophobic
amino acid side chains (see Figure 2b). Consequently,
lipophilic substituents were preferred, and certain steric
requirements existed to ll the lipophilic environment. This
observation was also captured by the steric elds of the
comparative molecular similarity index analysis (CoMSIA)
model used to analyze the ligand data. (Figure 3). Having no
substituent at the 5-position as in pyrazole 17a (R = H)
resulted in an essentially inactive compound and pinpointed the
necessity for further substitution in this region. Introduction of
substituted alkyl esters like 4 or 17b or isosteric lipophilic
ketones 17c, 17d, or 17e were eective to obtain signicant
binding anity and hPRP activity in the range of 2 M. In
P2Y12 binding assay using cell membranes from CHO cells with
recombinant expression of human P2Y12 receptors employing [33P]2MeS-ADP as the radioligand. bAntiagreggatory eect on ADPstimulated hPRP using a 96-well format. cIn parentheses: Antiagreggatory eect on ADP-stimulated hPRP according to the Born
method using single cuvettes.
contrast, the simple primary amide 17f or the acyclic tertiary

amide 17g did not improve the activity but were signicantly
less active. Interestingly, when cyclic pyrrolidine amides were
attached to the 5-position of the pyrazole instead (17h, 17i),
the most active compounds of this rst survey were obtained
reaching up to submicromolar potency in the hPRP assay [IC50
(hPRP): 1.37 and 0.9 M for 17h and 17i]. With this rst
evidence that cyclic amides could considerably enhance the
ligand anity and in particular the potency in the hPRP assay
and with the aim to avoid the imide functionality present in 17i,
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Table 2. Binding and hPRP Activity of Compounds Carrying Various Proline Variations
P2Y12 binding assay using cell membranes from CHO cells with recombinant expression of human P2Y12 receptors employing [33P]2-MeS-ADP as
the radioligand. bAntiagreggatory eect on ADP-stimulated hPRP using a 96-well format. cIn parentheses: Antiagreggatory eect on ADP-stimulated
hPRP according to the Born method using single cuvettes.
we turned our attention to proline derivatives, which appeared

to be closest analogues bearing a similar functionality.
Table 2 shows that even simple primary proline carboxamides like 18a essentially retain the activity as compared to
the initial pyrrolidine derivatives 17h and 17i. However, a

remarkable increase in binding anity and in particular hPRP
activity was observed with lipophilic secondary proline amides.
A stepwise extension of the lipophilic volume from methyl
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Table 3. Binding and hPRP Activity of Variations around the Piperazine Ethyl Carbamate
P2Y12 binding assay using cell membranes from CHO cells with recombinant expression of human P2Y12 receptors employing [33P]2-MeS-ADP as
the radioligand. bAntiagreggatory eect on ADP-stimulated hPRP using a 96-well format. cIn parentheses: Antiagreggatory eect on ADP-stimulated
hPRP according to the Born method using single cuvettes.
amide 18b over a linear ethyl amide 18c to a n-propyl amide

18d [IC50 (hPRP): 0.058 M] strongly increased the activity.
Successive branching of the terminal lipophilic amide from the
isopropyl amide 18e to the spherical tert-butyl amide 18f
further enhanced the activity [IC50 (hPRP): 0.040 and 0.017
M]. Notably, the cyclopropyl amide 18g [IC50 (hPRP): 0.013
M] was even more potent as compared to the corresponding
isosteric acyclic isopropyl amide 18e. Interestingly, the (R)enantiomer 18h addressing the opposite trajectory of (S)enantiomer 18g showed a more than 10-fold drop of binding
anity as compared to 18g, suggesting a highly specic
hydrophobic interaction of 18g with the receptor. Further
extension of the ring size from a cyclopropyl amide (18g) to a

cyclobutyl amide (18i) or cyclopentyl amide (18j) unveiled
one of the most potent substitution patterns of the present
study, displaying optimal binding anity and hPRP activity in
the nanomolar range [Ki (P2Y12): 7.3 and 20 nM; IC50 (hPRP):
0.025 and 0.013 M]. This very subtle interaction is further
illustrated by the compounds 18k18m. Small variations from
the optimal presentation of the cyclobutyl moiety like a proline
N-methyl-cyclobutyl amide 18k, a methylene-linker extension
in 18l, or the ring-contracted proline analogue, azetidine 18m,
resulted in moderate loss of binding anity and a moderate to
signicant drop of hPRP activity. Stronger deviations from the
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corresponding 2- or 3-methyl-substituted piperazines 19mp

were investigated. Interestingly, a pronounced inuence of the
position and absolute conguration of the methyl group was
observed. Among the variations 19m to 19p, only the (3R)congured stereoisomer 19m was able to restore the activity of
the nonsubstituted piperazine derivative 18i in the binding and
hPRP assay. All other variations were less active. This result is
indicative for the considerable spatial restrictions and
conformational preferences for the receptorligand interaction.
Previous studies11a,c,16,18 suggested an indispensable role of
the -carboxylic acid moiety of the central glutamic acid for
binding anity by a strong putative salt bridge interaction with
Lys7.35 and Arg6.55 at the ADP binding pocket (see also
Figure 2). Despite these putative prerequisites, we hypothesized
that the remarkably strong receptor interaction of the N-acetyl(S)-proline cyclobutyl amide-substituted pyrazolone backbone
would allow us to vary the central amino acid region without a
serious loss of activity. Table 4 shows the set of variations made
to probe the signicance of the side chain of the glutamic acid
linker and to evaluate the spatial and electrostatic environment.
Examination of compounds bearing polar amino acid side
chains like sulfonamide 20a or 20b did not reveal any superior
motif than the glutamic acid moiety in 18i. None of these
variations showed hPRP IC50 values better than 100 nM.
Nonetheless, the detected binding anities around 100 nM for
those ligands indicated that there were no severe unfavorable
interactions present. Consequently, when the carboxylic acid
portion in compounds 18i and 19k was replaced by the
nonclassical bioisostere tetrazole, equally potent compounds
20c and 20d were obtained [Ki (P2Y12): 5.0 and 1.5 nM; IC50
(hPRP): 0.040 and 0.050 M]. In agreement with our docking
hypothesis, introduction of a basic ethyl amine side chain 20e
led to a signicant drop of activity, which could be partially
recovered by acetylation (20f), suggesting that the amino acid
side chain is accommodated in a positively charged environment of the receptor. Notably, the simple noncharged C2
serine homologue 20g turned out to be very active [Ki (P2Y12):
23 nM; IC50 (hPRP): 0.210 M]. Further chain elongation led
to the corresponding C3 serine homologue 20h, which
exhibited an even higher binding anity and a hPRP activity
in the low nanomolar range [Ki (P2Y12): 6.5 nM; IC50 (hPRP):
0.092 M]. However, commencing spatial restrictions became
evident with the corresponding longer hydroxyl derivative 20i,
which was found to be inferior in terms of both binding anity
and hPRP activity. Deletion of any polarity by introducing
linear and branched lipophilic amino acids of dierent length
and extension (20jm) showed in general lower binding
anities and much lower hPRP activities, supporting the
hypothesis that there are no favorable hydrophobic contacts at
this site of the receptor binding pocket and that competing
plasma protein binding due to the increased lipophilicity is
likely. Unexpectedly, replacement of alanine (20m) by glycine
(20n) led to a more than 20-fold increase in hPRP activity
through sole omission of a single methyl group [Ki (P2Y12):
111 vs 107 nM; IC50 (hPRP): 0.090 vs 2.21 M]. The
corresponding n-butyl carbamate 20o was found to be even
more potent in the binding assay and in the hPRP assay, closely
reaching the potency of the analogues bearing the glutamic acid
linker [Ki (P2Y12): 7.7 nM; IC50 (hPRP): 0.030 M]. One
might hypothesize that in the absence of the glutamic acid or
any other side chain, the interaction with the basic residues is
established via one or more water molecules. However, this
result was highly unexpected as in related series described by
optimal trajectorial angle presenting the cyclobutyl carboxamide moiety as in compounds 18n or 18o turned out to be
deleterious for the activity [Ki (P2Y12): 238 and 613 nM; IC50
(hPRP): 3.13 and 2.94 M]. Next, we explored compounds
bearing a cyclic amide variation of increasing ring size. Again,
even small, incremental changes from the moderately active
pyrrolidine carboxamide 18p to the piperidine carboxamide
18q were sucient to obtain an acceptable binding anity and
potency in hPRP [Ki (P2Y12): 219 vs 56 nM; IC50 (hPRP):
0.60 vs 0.070 M]. The attempted bioisosteric replacement of
the carboxamide group with an imidazole moiety resulted in the
reasonably active compound 18r. Notably, the profound impact
of the lipophilic interaction with the receptor at this region was
also demonstrated when the entire alkyl carboxamide was
omitted and replaced by a phenyl ring. Despite the relatively
large structural change, this compound 18s displayed an
unexpectedly high binding anity and hPRP activity as
compared to several other more conservative variations of the
proline cyclobutyl amide motif [Ki (P2Y12): 33 nM; IC50
(hPRP): 0.29 M].
These results indicate that the 5-position of the pyrazole ring
directs the substituent to a lipophilic pocket, which exhibits a
steric connement, as illustrated by the signicant drop of
activity, if the ideal geometry is violated. A more concise
structure-based rationalization of the observed SAR is provided
in the section Development of 3D QSAR Models.
The results in Table 2 established the structural requirements
for the decoration of the 5-position of the phenylpyrazole with
the N-acetyl-(S)-proline cyclobutyl amide residue as the
optimal substituent. We next turned our attention toward a
potential optimization of the piperazine carbamate portion of
our preliminary lead compound (Table 3). Our docking
hypothesis (Figure 1) proposed that this substituent points to
the extracellular site of the receptor and establishes interactions
with residues from the ECs. Because we considered the
accuracy of our protein model in this region to be very low, a
reliable structure-based rationalization of the piperazine
carbamate SAR was not feasible. Nevertheless, the preference
for lipophilic residues of a certain length (e.g., butyl
substituents) was captured by the steric elds of the CoMSIA
model (see Figure 3). Introducing a 4-amino piperidine group
as a piperazine homologue resulted in the considerably less
active compound 19a (Table 3). Interestingly, 19b incorporating the inverse 4-amino piperidine spacer exhibited a higher
binding anity to the receptor and had a much higher activity
in hPRP than 19a [Ki (P2Y12): 22 vs >5000 nM; IC50 (hPRP):
0.055 vs >5.0 M]. The same trend was also observed for the
related analogues, the corresponding 3-amino pyrrolidine
compounds 19c and 19d, which conrmed that a C-terminal
attachment of the glutamic acid to a secondary amine is crucial
for high receptor anity. Any eort to replace the alkyl
carbamate unit by more rigid N-aryl piperazines like in 19e or
N-acyl piperazines like in 19g was unsuccessful and did not lead
to compounds of higher binding anity or hPRP activity. In
contrast, very conservative variations of the alkyl carbamate
moiety turned out to be most eective. Simple alkyl extension
from ethyl (18i) to n-propyl (19j) and n-butyl (19k) provided
derivatives that showed the highest binding anities and an
extraordinarily good hPRP activity [Ki (P2Y12): 3.1 and 0.8 nM;
IC50 (hPRP): 0.021 and 0.019 M]. An equal potency was
found for the cyclobutyl derivative 19l [Ki (P2Y12): 5.0 nM;
IC50 (hPRP): 0.014 M]. In an attempt to rigidify and restrict
the conformational exibility of the piperazine spacer, the
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others,11a,c the corresponding glycine analogues were found to

be inactive in the hPRP assay. Therefore, it has to be
anticipated that the strong impact of the cyclic amides at the 5position of the pyrazole on the hPRP activity apparently
outbalances the absence of the carboxyl group of the glutamic
acid moiety, which usually is required to obtain good activities
in both binding and hPRP assays.
Selected compounds were further evaluated to elucidate the
origin of the observed very high inhibitory activity in the hPRP
assay as compared to the initial lead 4 (Table 5). To estimate
Table 4. Binding and hPRP Activity of Compounds with

Variations around the Central Amino Acid Moiety
Table 5. Activity and Selectivity Data of Selected

Compounds Using P2Y1 Cell Membranes and IPs
Stimulated with Dierent Agonists
compds
Ki (P2Y12)a
Ki (P2Y1)b
IC50 (IP, ADP)c
IC50 (hPRP, ADP)d
IC50 (IP, col)e
IC50 (IP, thr)f
18i
20n
20o
139 nM
>20 M
0.701 M
2.93 M
>2 M
>2 M
7.3 nM
>20 M
0.007 M
0.009 M
>2 M
>2 M
107 nM
ND
0.099 M
0.100 M
>2 M
>2 M
7.7 nM
>20 M
0.022 M
0.046 M
>2 M
>2 M
recombinant expression of human P2Y12 receptors employing [33P]2MeS-ADP as the radioligand. bP2Y1 binding assay using cell
membranes from CHO cells with recombinant expression of human
P2Y1 receptors employing [33P]2-MeS-ADP as the radioligand.
c
Antiagreggatory eect on ADP-stimulated human IPs according to
the Born method using single cuvettes. dAntiagreggatory eect on
ADP-stimulated hPRP according to the Born method using single
cuvettes. eAntiagreggatory eect on collagen (col)-stimulated human
IP according to the Born method using single cuvettes. fAntiagreggatory eect on thrombin (thr)-stimulated human IP according to the
Born method using single cuvettes.
the contribution of a reduced association of the compounds to

plasma proteins, the antiaggregatory activity with isolated
platelets (IPs) preparations was determined to avoid additional
blood components interfering with the inhibitory eects. As
expected from the binding anity and hPRP data, lead 4 was
also the least active compound on IPs. Moreover, a comparison
of the respective activity in hPRP reveals that lead compound 4
exhibits the highest loss of antiaggregatory activity in hPRP
with a 4-fold shift in the presence of plasma proteins [IC50 (IP):
0.701 M vs IC50 (hPRP): 2.93 M]. In contrast, the
compounds 18i, 20n, and 20o, bearing the N-acetyl-(S)-proline
cyclobutyl amide moiety, showed only a moderate shift in
activity in hPRP as compared to IP, thus indicating that the
association to plasma proteins does not impair the antiaggregatory activity to a signicant extent. To exclude that the
observed strong antagonistic action of these compounds
originates from the interference of the ligands with other
activation pathways, IPs were stimulated using Col and Thr as
agonists in the presence of the 4, 18i, 20n, and 20o. Neither the
Col- nor the Thr-stimulated aggregation was aected at
relevant concentrations by 4, 18i, 20n, and 20o.9,19 In addition,
these compounds were highly selective versus the P2Y1
receptor, thus excluding any synergistic interaction and
contribution of this receptor. These results clearly indicate
that the observed strong inhibition of the ADP-mediated
platelet aggregation in hPRP is due to the specic antagonistic
action on the P2Y12 receptor. Moreover, apart from the high
functional activity shown in IP, the reduced plasma protein
recombinant expression of human P2Y12 receptors employing [33P]2MeS-ADP as the radioligand. bAntiagreggatory eect on ADPstimulated hPRP using a 96-well format. cIn parentheses: Antiagreggatory eect on ADP-stimulated hPRP according to the Born
method using single cuvettes.
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binding appears to contribute to the observed high antagonistic

activity in hPRP.
The potential for the further development of these
compounds was underscored by the evaluation of the
antiaggregatory activity in an ex-vivo dog model.9,20 For
example, when 20o was orally administered with 10 mg/kg a
sustained antiaggregatory eect with 73% inhibition after 24 h
[whole blood platelet aggregation (impedance), 2.5 M ADP,
area under curve (AUC mean)] was observed.
4. DEVELOPMENT OF 3D QSAR MODELS

Because the binding hypotheses provided in this publication
were based on docking modes into homology models and not
on high-resolution crystal structures, we generated multiple
ligand alignments for the establishment of 3D QSAR
[comparative molecular eld analysis (CoMFA) and CoMSIA]
models to reveal essential features for activity. Starting with the
docking mode of 4 (Figure 2) as a reference, all remaining
compounds were consistently aligned with our multiple ligand
alignment approach Multiple Alignments by ROCS-based
Similarity (MARS).21 On the basis of this alignment,
statistically good CoMFA (qLOO2 = 0.60, qLNO2 = 0.59 for
four partial least-squares components) and CoMSIA (qLOO2 =
0.63, qLNO2 = 0.62 for four partial least-squares components)
models were obtained. The steric and electrostatic std*coe
elds of the CoMSIA model are shown in Figure 3 with 4 [Ki
(P2Y12): 139 nM]. Green contours indicate regions where
steric bulk is favorable for P2Y12 activity, whereas yellow
contours highlight regions where substituents are detrimental
for activity.
Because the reference conformation for the multiple ligand
alignment was based on a docking hypothesis in the P2Y12
receptor, the contour elds can be analyzed in the context of
the binding site (model) environment. Indeed, the maps are
consistent with steric and electrostatic requirements of the
modeled P2Y12 binding site. For example, the carboxy group of
the central glutamic acid of the initial lead 4 is located close to a
region (indicated by a red contour) where negatively charged
substituents are favorable for activity due to electrostatic
interactions to the cationic amino acids Arg6.55 and Lys7.35.
Figure 3 panels ad illustrate the SAR of the substituents at the
5-position of the pyrazole moiety. The benecial eect of
lipophilic substituents at the 5-position is visualized in Figure
3b,c by the steric elds (indicated by a green contour). For
example, the propyl side chain of 18d [Ki (P2Y12): 66 nM] is
located in a green contour eld, where additional steric bulk is
favorable for anity. This is in contrast to the methyl
substituent of 18b, which shows a considerably lower activity
[Ki (P2Y12): 536 nM]. Figure 3d explains the congurational
dependence of the activity for the proline stereoisomers 18g
and 18h (Table 2). Whereas the (S)-congured stereoisomer
18g [Ki (P2Y12): 105 nM] shows an optimal interaction with
the modeled binding site, the (R)-congured stereoisomer 18h
[Ki (P2Y12): 958 nM] clashes with the surrounding protein
residues, resulting in a less favorable overall t of the molecule
in the receptor binding site.
Figure 3. (a) Shows steric and electrostatic std*coe contour maps

from the CoMSIA model with our initial lead compound 4 [Ki
(P2Y12), 139 nM]: Green contours refer to regions where additional
steric bulk is favorable for anity, while yellow contours indicate
disfavored areas. Red contours indicate regions where negatively
charged substituents are favorable. (b) The same as panel a with all 63
compounds aligned. (c) The same as panel a with 18d [Ki (P2Y12), 66
nM]. (d) The same as panel a with 18g [molecule shown in green; Ki
(P2Y12), 105 nM] and 18h [yellow; Ki (P2Y12), 958 nM].
residues attached to the 5-O-position of the pyrazolone like the

N-acetyl-(S)-proline cyclobutyl amide group accumulated
strong evidence that these moieties address a distinct binding
pocket of the receptor. Addressing this binding pocket resulted
in not only an increase in binding anity but also a dramatic
increase of functional hPRP activity, a recognized parameter for
the antithrombotic activity in the clinic, up to the low
nanomolar range. Surprisingly, revisiting the structural requirements of the piperazinyl ethyl carbamate and the central
glutamic acid moiety revealed that the charged glutamate
residue could be replaced by a glycine. This was highly
unexpected, as the glutamic residue had been previously
considered to form an indispensable strong interaction with the
basic residues Arg6.55 and Lys7.35 of the P2Y12 receptor.
Further investigations using IPs and other agonists revealed a
reduced association to plasma proteins of these compounds as
compared to the initial lead 4 and excluded the interference of
the antagonists with other signicant activation pathways.
These results underscore the key role played by the high anity
N-acetyl-(S)-proline cyclobutyl amide ligand moiety, in
determining not only the high binding anity but also the
strong functional hPRP activity. Exploiting this behavior may
potentially allow the generation of other highly potent P2Y12
antagonists by using other N-acetyl-(S)-proline cyclobutyl
amide ligandscaold combinations.
5. CONCLUSION
We have developed a series of highly active P2Y12 antagonists
based on a phenylpyrazolone scaold. Investigation of the
structural requirements at three dierent regions generated a
set of structural diverse P2Y12 antagonists. Notably, noncharged
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7.4 Hz, 2H), 3.52 (m, 4H), 3.39 (m, 4H), 2.27 (m, 2H), 1.80 (m, 1H),
1.67 (m, 1H), 1.39 (s, 9H), 1.19 (t, J = 7.4 Hz, 3H). tR (method j):
1.52 min. MS (ES) m/z: 478.3 (MH+).
6.1.1.2. 4-((S)-2-Amino-4-tert-butoxycarbonyl-butyryl)-piperazine-1-carboxylic Acid Ethyl Ester (7). To a solution of 4-((S)-2benzyloxycarbonylamino-4-tert-butoxycarbonyl-butyryl)-piperazine-1carboxylic acid ethyl ester (20.9 g, 43.9 mmol) in ethanol (120 mL)
was added Pd/C (2.0 g, 10% w/w), and the suspension was stirred
under an atmosphere of hydrogen (3 bar) for 12 h. The reaction
mixture was ltrated over a plug of Celite, washed with ethanol, and
concentrated to give the pure product 7 (13.6 g, 89%) as a colorless
oil, which was not further puried. 1H NMR (DMSO-d6): 4.06 (q, J
= 7.4 Hz, 2H), 3.57 (m, 1H), 3.49 (m, 4H), 3.37 (m, 4H), 2.30 (m,
2H), 1.65 (m, 2H), 1.39 (s, 9H), 1.19 (t, J = 7.4 Hz, 3H). tR (method
j): 0.82 min. MS (ES) m/z: 344.3 (MH+).
6.1.2. 5-Carboxymethoxy-1-phenyl-1H-pyrazole-3-carboxylic
Acid Ethyl Ester (9). 6.1.2.1. 5-Benzyloxycarbonylmethoxy-1phenyl-1H-pyrazole-3-carboxylic Acid Ethyl Ester. To a solution of
5-hydroxy-1-phenyl-1H-pyrazole-3-carboxylic acid ethyl ester (8)
(10.0 g, 43.1 mmol) in DMF (100 mL) were added benzyl
bromoacetate (7.1 mL, 43.1 mmol) and cesium carbonate (24.0 g,
86.1 mmol). After it was stirred for 16 h at room temperature, the
suspension was ltered, washed with DMF, and concentrated. The
residue was taken up with ethyl acetate and extracted with aqueous
LiCl (4% w/w). Evaporation of the solvent yielded the crude product
as a yellow oil (15.5 g, 95%), which was used in the subsequent
debenzylation reaction. tR (method j): 1.64 min. MS (ES) m/z: 381.2
(MH+).
6.1.2.2. 5-Carboxymethoxy-1-phenyl-1H-pyrazole-3-carboxylic
Acid Ethyl Ester (9). To a solution of 5-benzyloxycarbonylmethoxy1-phenyl-1H-pyrazole-3-carboxylic acid ethyl ester (15.5 g, 40.8 mmol)
in ethyl acetate (100 mL) was added Pd/C (1 g, 10%), and the
resulting suspension stirred under an atmosphere of hydrogen (3 bar)
for 5 h. The reaction mixture was ltered over a plug of Celite and
washed with ethyl acetate and methanol to give the crude product 9 as
colorless platelets (11.5 g, 98%) after evaporation of the solvents. 1H
NMR (DMSO-d6): 13.28 (s br, 1H), 7.74 (d, J = 7.9 Hz, 2H), 7.54
(t, J = 7.9 Hz, 2H), 7.42 (t, J = 7.9 Hz, 1H), 6.42 (s, 1H), 4.93 (s, 2H),
4.29 (q, J = 7.0 Hz, 2H), 1.30 (t, J = 7.0 Hz, 3H). tR (method j): 1.11
min. MS (ES) m/z: 291.2 (MH+).
6.1.3. Preparation of 5-[2-((S)-2-cyclobutylcarbamoyl-pyrrolidin1-yl)-2-oxo-ethoxy]-1-phenyl-1H-pyrazole-3-carboxylic Acid (10).
6.1.3.1. 5-[2-((S)-2-Benzyloxycarbonyl-pyrrolidin-1-yl)-2-oxoethoxy]-1-phenyl-1H-pyrazole-3-carboxylic Acid Ethyl Ester. To a
solution of 5-carboxymethoxy-1-phenyl-1H-pyrazole-3-carboxylic acid
ethyl ester (9) (9.5 g, 32.8 mmol) in DMF (100 mL) were added
HOBt (5.0 g, 32.8 mmol), EDC (6.3 g, 32.8 mmol), and DIPEA (10.9
mL, 65.7 mmol). After 5 min, L-proline benzyl ester hydrochloride (7.9
g, 32.8 mmol) was added, and the resulting solution was stirred for 16
h. The reaction mixture was concentrated, dissolved in dichloromethane, and extracted with aqueous LiCl (4% w/w) and saturated
NaHCO3. The crude product obtained after evaporation of the solvent
was puried by ash chromatography on silica using ethyl acetate/
heptane 1:1 as an eluent to give the product as colorless foam (13.2 g,
84%). tR (method j): 1.55 min. MS (ES) m/z: 478.3 (MH+).
6.1.3.2. 5-[2-((S)-2-Carboxy-pyrrolidin-1-yl)-2-oxo-ethoxy]-1-phenyl-1H-pyrazole-3-carboxylic Acid Ethyl Ester. To a solution of 5-[2((S)-2-benzyloxycarbonyl-pyrrolidin-1-yl)-2-oxo-ethoxy]-1-phenyl1H-pyrazole-3-carboxylic acid ethyl ester (13.2 g, 27.6 mmol) in ethyl
acetate (100 mL) was added Pd/C (1 g, 10%), and the resulting
suspension was stirred under an atmosphere of hydrogen (3 bar) for 6
h. The reaction mixture was ltered over a plug of Celite and washed
with ethanol to give the crude product as colorless foam (8.9 g, 70%)
after evaporation of the solvents. 1H NMR (DMSO-d6): 12.61 (s br,
1H), 7.83 (d, J = 8.4 Hz, 2H), 7.57 (t, J = 7.8 Hz, 2H), 7.45 (t, J = 7.8
Hz, 1H), 6.48 (s, appears as 2 rotamers, 1H), 5.12 (s, 2H), 4.32 (m,
3H), 3.55 (m, 2H), 2.15 (m, 1H), 1.89 (m, 3H), 1.28 (t, J = 7.0 Hz,
3H). tR (method j): 1.10 min. MS (ES) m/z: 388.2 (MH+).
6.1.3.3. 5-[2-((S)-2-Cyclobutylcarbamoyl-pyrrolidin-1-yl)-2-oxoethoxy]-1-phenyl-1H-pyrazole-3-carboxylic Acid Ethyl Ester. To a
solution of 5-[2-((S)-2-carboxy-pyrrolidin-1-yl)-2-oxo-ethoxy]-1-phe-
6. EXPERIMENTAL SECTION
6.1. Chemistry. Solvents and other reagents were used as received
without further purication. Normal phase column chromatography
was carried out on Merck silica gel 60 (230400 mesh). Reversed
phase high-pressure chromatography was conducted on an Agilent
1100 instrument using an Agilent Prep. C18 column (10 m, 30 mm
250 mm). Thin-layer chromatography (TLC) was carried out on
TLC aluminum sheets with silica gel 60F254 from Merck. Varying
ratios of acetonitrile and 0.1% triuoroacetic acid in water were used as
solvent systems. NMR spectra were recorded in CD3OD, CDCl3, or
DMSO-d6 either on a Bruker DRX 400 or a Bruker FN-ARX 500.
Chemical shifts are reported as values from an internal
tetramethylsilane standard. All nal compounds were puried by
normal phase or reversed phase chromatography. Purity and
characterization of compounds were established by a combination of
analytical HPLC, LC-MS, and NMR analytical techniques. All tested
compounds were found to be >95% pure by analytical HPLC and LCMS analysis.
LC-MS analyses were performed with an Agilent HPLC Series
1100. The following HPLC methods were used to obtain the reported
retention times. Method a: YMC Jsphere column 33 mm 2 mm; 4
M; ow rate, 1.0 mL/min. Gradient: water + 0.05% triuoroacetic
acid:acetonitrile + 0.05% triuoroacetic acid: 98 (1 min) to 95:5 (5.0
min) to 95:5 (6.25 min); Waters LCT-MUX, ESI+ mode. Method b:
YMC Jsphere column 33 mm 2 mm; 4 m; ow rate, 1.0 mL/min.
Gradient: water + 0.05% triuoroacetic acid:acetonitrile + 0.05%
triuoroacetic acid 95:5 (0 min) to 5:95 (3.7 min); Waters LCTMUX, ESI+ mode. Method c: Waters XBridge C18 column 4.6 mm
50 mm; 2.5 m; ow rate, 1.3 mL/min. Gradient: water + 0.1% formic
acid:acetonitrile + 0.08% formic acid 97:3 (0 min) to 40:60 (3.5 min)
to 2:98 (4 min) to 2:98 (5 min) to 97:3 (5.2 min) to 97:3 (6.5 min);
Waters Quattro Ultima, ESI+ mode. Method d: Waters XBridge C18
column 4.6 mm 50 mm; 2.5 m; ow rate, 1.3 mL/min. Gradient:
water + 0.05% triuoroacetic acid:acetonitrile + 0.05% triuoroacetic
acid 95:5 (0 min) to 95:5 (0.3 min) to 5:95 (3.5 min) to 5:95 (4 min);
Waters LCT, ESI+ mode. Method e: YMC Jsphere column 33 mm 2
mm; 4 m; ow rate, 1.3 mL/min. Gradient: water + 0.05%
triuoroacetic acid:acetonitrile + 0.05% triuoroacetic acid 95:5 (0
min) to 5:95 (2.5 min) to 95:5 (3.2 min); Waters LCT, ESI+ mode.
Method f: YMC Jsphere column 33 mm 2 mm; 4 m; ow rate, 1.0
mL/min. Gradient water + 0.05% triuoroacetic acid:AcN + 0.05%
triuoroacetic acid 95:5 (0 min) to 5:95 (3.7 min); Waters LCTMUX, ESI+ mode. Method g: YMC Jsphere column 33 mm 2 mm;
4 m; ow rate, 1.3 mL/min. Gradient: water + 0.05% formic
acid:acetonitrile + 0.05% formic acid 95:5 (0 min) to 95:5 (0.5 min) to
5:95 (3.5 min) to 5:95 (4 min); Waters LCT, ESI+ mode. Method h:
YMC Jsphere column 33 mm 2 mm; 4 m; ow rate, 1.3 mL/min.
Gradient: water + 0.05% formic acid:acetonitrile + 0.05% formic acid
95:5 (0 min) to 5:95 (2.5 min) to 5:95 (3 min); Waters LCT, ESI+
mode. Method i: YMC Jsphere column 33 mm 2 mm; 4 m; ow
rate, 1.3 mL/min. Gradient: water + 0.1% formic acid:acetonitrile +
0.08% formic acid 95:5 (0 min) to 5:95 (2.5 min); Waters Quattro
Ultima, ESI+ mode. Method j: YMC Jsphere column 20 mm 2 mm;
4 m; ow rate, 1.0 mL/min. Gradient: water + 0.05% triuoroacetic
acid:acetonitrile + 0.05% triuoroacetic acid 94:6 (0 min) to 5:95 (2.4
min) to 94:6 (2.45 min); Agilent series 1100 MSD, ESI+ mode.
6.1.1. 4-((S)-2-Amino-4-tert-butoxycarbonyl-butyryl)-piperazine1-carboxylic Acid Ethyl Ester (7). 6.1.1.1. 4-((S)-2-Benzyloxycarbonylamino-4-tert-butoxycarbonyl-butyryl)-piperazine-1-carboxylic
Acid Ethyl Ester. To a solution of (S)-2-benzyloxycarbonylaminopentanedioic acid 5-tert-butyl ester (5) (15.0 g, 44.5 mmol) in DMF
(75 mL) were added 1-ethoxycarbonylpiperazine (6) (6.9 mL, 44.5
mmol), N-ethylmorpholine (22.6 mL, 177.8 mmol), and TOTU (14.6
g, 44.5 mmol). After it was stirred for 12 h, the solution was diluted
with ethyl acetate and subsequently washed with aqueous LiCl (4% w/
w) and saturated aqueous NaHCO3. The crude product (20.9 g, 99%)
obtained after evaporation of the solvent was directly subjected to the
subsequent deprotection reaction without further purication. 1H
NMR (DMSO-d6): 7.52 (d, J = 8.3 Hz, 1H), 7.34 (m, 5H), 5.04 (d, J
= 12.6 Hz, 1H), 4.99 (d, J = 12.6 Hz, 1H), 4.47 (m, 1H), 4.06 (q, J =
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nyl-1H-pyrazole-3-carboxylic acid ethyl ester 84.5 g, 11.6 mmol) in

DMF (105 mL) were added HATU (4.4 g, 11.6 mmol) and DIPEA
(1.9 mL, 11.6 mmol). After 10 min, cyclobutylamine (1.0 mL, 11.6
mmol) was added, and after 30 min, the reaction mixture was
concentrated. The residue was taken up in dichloromethane and
extracted with aqueous LiCl (4% w/w), aqueous HCl (0.1 M), and
saturated NaHCO3. The crude product obtained after evaporation of
the solvent was puried by ash chromatography on silica using ethyl
acetate/heptane 4:1 as the eluent to give the title compound as a
colorless oil (3.2 g, 62%). tR (method j): 1.26 min. MS (ES) m/z:
441.3 (MH+).
6.1.3.4. 5-[2-((S)-2-Cyclobutylcarbamoyl-pyrrolidin-1-yl)-2-oxoethoxy]-1-phenyl-1H-pyrazole-3-carboxylic Acid (10). To a solution
of 5-[2-((S)-2-cyclobutylcarbamoyl-pyrrolidin-1-yl)-2-oxo-ethoxy]-1phenyl-1H-pyrazole-3-carboxylic acid ethyl ester (3.2 g, 7.2 mmol)
in THF (40 mL) and water (5 mL) was added NaOH (340 mg, 8.5
mmol) portionwise at 0 C. After 4 h, the solution was neutralized
with Amberlite IR-120 ion-exchange resin, ltered, and washed with
methanol. After evaporation of the solvents, the product 10 was
obtained as a yellow gum (3.7 g, 77%). 1H NMR (DMSO-d6): 8.00
(d, appears as 2 rotamers, J = 7.5 Hz, 1H), 7.81 (d, J = 8.7 Hz, 2H),
7.48 (t, J = 8.7 Hz, 2H), 7.33 (t, J = 8.7 Hz, 1H), 6.24 (s, appears as 2
rotamers, 1H,), 4.99 (s, 2H), 4.22 (dd, J = 8.6 Hz, J = 3.5 Hz, 1H),
4.14 (m, 1H), 3.56 (m, 1H), 3.47 (m, 1H), 2.201.54 (several
multiplets, 10H). tR (method j): 1.04 min. MS (ES) m/z: 413.2 (MH
+).
6.1.4. Representative Procedure: Preparation of 4-{(S)-4-Carboxy2-[(5-hydroxy-1-phenyl-1H-pyrazole-3-carbonyl)-amino]-butyryl}piperazine-1-carboxylic Acid Ethyl Ester (17a). 6.1.4.1. 4-{(S)-4-tertButoxycarbonyl-2-[(5-hydroxy-1-phenyl-1H-pyrazole-3-carbonyl)amino]-butyryl}-piperazine-1-carboxylic Acid Ethyl Ester. To a
solution of 1-phenyl-3-carboxy-5-pyrazolone 11 (2 g, 35.2 mmol)
and 4-((S)-2-amino-4-tert-butoxycarbonyl-butyryl)-piperazine-1-carboxylic acid ethyl ester (7) (12.1 g, 35.2 mmol) in DMF (100 mL),
HOBt (5.4 g, 35.2 mmol) and EDC (6.7 g, 35.2 mmol) were added,
and the reaction mixture was stirred for 12 h at room temperature.
Then, the reaction mixture was diluted with ethyl acetate and
subsequently extracted with aqueous LiCl (4% w/w), aqueous HCl
(0.1 M), and aqueous NaHCO3. The organic layer was dried over
MgSO4, and the solvent was removed under reduced pressure to yield
the product (14.8 g, 80%), which was directly subjected to the
subsequent deprotection reaction.
6.1.4.2. 4-{(S)-4-Carboxy-2-[(5-hydroxy-1-phenyl-1H-pyrazole-3carbonyl)-amino]-butyryl}-piperazine-1-carboxylic Acid Ethyl Ester
(17a). To a solution of 4-{(S)-4-tert-butoxycarbonyl-2-[(5-hydroxy-1phenyl-1H-pyrazole-3-carbonyl)-amino]-butyryl}-piperazine-1-carboxylic acid ethyl ester (50 mg, 0.09 mmol) in dichloromethane (2 mL),
triuoroacetic acid (0.13 mL) was added. After it was stirred for 4 h,
the reaction mixture was concentrated, and the crude product obtained
was puried by preparative reversed-phase HPLC, eluting with a
gradient of acetonitrile in water (+0.01% triuoroacetic acid). After
lyophilization, the product 17a (9 mg, 20%) was obtained as a white
solid. 1H NMR (DMSO-d6): 12.05 (s br, 1H), 8.09 (d, J = 7.8 Hz,
1H), 7.78 (d, J = 8.1 Hz, 2H), 7.50 (t, J = 7.8 Hz, 2H), 7.35 (t, J = 7.1
Hz, 1H), 5.92 (s, 1H), 4.94 (dt, J = 4.5 Hz, J = 8.8 Hz, 1H), 4.06 (q, J
= 6.8 Hz, 2H), 3.46 (m, 8H), 2.29 (m, 2H), 1.96 (m, 1H), 1.80 (m,
1H), 1.19 (t, J = 6.8 Hz, 3H). tR (method e): 1.39 min. MS (ES) m/z:
474.2 (MH+).
6.1.5. Representative Procedure: Preparation of 4-((S)-4-Carboxy2-{[5-(1-ethoxycarbonyl-cyclobutoxy)-1-phenyl-1H-pyrazole-3-carbonyl]-amino}-butyryl)-piperazine-1-carboxylic Acid Ethyl Ester (4).
To a solution of 4-{(S)-4-tert-butoxycarbonyl-2-[(5-hydroxy-1-phenyl1H-pyrazole-3-carbonyl)-amino]-butyryl}-piperazine-1-carboxylic acid
ethyl ester (1.50 g, 2.8 mmol) in DMF (20 mL), ethyl 1bromocyclobutanecarboxylate (1.22 g, 5.7 mmol) and cesium
carbonate (1.85 g, 5.66 mmol) were added. The reaction mixture
was stirred for 2 h at 100 C, then cooled to room temperature, and
diluted with water. The solution was extracted with dichloromethane,
and the organic layer was washed with water and dried over MgSO4.
After the solvents were removed under reduced pressure, the residue
was puried by ash chromatography on silica using a heptane/ethyl
acetate gradient to give the corresponding O-alkylation product (860

mg, 46%), which was dissolved in dichloromethane (5 mL) and stirred
in the presence of of triuoroacetic acid (0.4 mL). After they were
stirred for 4 h at room temperature, the solvents were removed under
reduced pressure, and the residue was puried by ash chromatography on silica using a dichloromethane/ethanol gradient. After
lyophilization, the product 4 (320 mg, 40%) was obtained as a white
solid. 1H NMR (DMSO-d6): 8.20 (d, J = 8.0 Hz, 1H), 7.76 (d, J =
9.0 Hz, 2H), 7.55 (t, J = 8.0 Hz, 2H), 7.42 (t, J = 7.3 Hz, 1H), 5.84 (s,
1H), 4.93 (m, 1H), 4.17 (q, J = 6.9 Hz, 2H), 4.05 (q, J = 7.0 Hz, 2H),
3.46 (m, 8H), 2.68 (m, 2H), 2.50 (m, 2H), 2.25 (m, 2H), 1.86 (m,
4H), 1.19 (t, J = 6.8 Hz, 3H), 1.12 (t, J = 6.8 Hz, 3H). tR (method f):
2.08 min. MS (ES) m/z: 600.3 (MH+).
6.1.6. Representative Procedure: Preparation of 4-[(S)-4-Carboxy2-({5-[2-((R)-3-hydroxy-pyrrolidin-1-yl)-2-oxo-ethoxy]-1-phenyl-1Hpyrazole-3-carbonyl}-amino)-butyryl]-piperazine-1-carboxylic Acid
Ethyl Ester (17h) via Route 1 (Scheme 2). 6.1.6.1. 4-{(S)-2-[(5Benzyloxycarbonylmethoxy-1-phenyl-1H-pyrazole-3-carbonyl)amino]-4-tert-butoxycarbonyl-butyryl}-piperazine-1-carboxylic
Acid Ethyl Ester. To a solution of 4-{(S)-4-tert-butoxycarbonyl-2-[(5hydroxy-1-phenyl-1H-pyrazole-3-carbonyl)-amino]-butyryl}-piperazine-1-carboxylic acid ethyl ester (14.5 g, 27.3 mmol) in DMF (110
mL) were added benzyl bromoacetate (4.5 mL, 27.3 mmol) and
cesium carbonate (17.8 g, 54.7 mmol). After it was stirred at room
temperature for 12 h, the solution was reduced to a volume of 50 mL,
diluted with ethyl acetate (400 mL), and extracted with aqueous LiCl
(4% w/w). The crude product obtained after evaporation of the
solvent was puried by ash chromatography on silica eluting with a
gradient of n-heptane/ethyl acetate to give the title compound as a
yellowish amorphous solid (13.2 g, 71%). 1H NMR (DMSO-d6):
8.14 (d, J = 8.2 Hz, 1H), 7.77 (d, J = 8.0 Hz, 2H), 7.51 (t, J = 8.0 Hz,
2H), 7.40 (t, J = 8.0 Hz, 1H), 7.36 (m, 5H), 6.42 (s, 1H), 5.21 (s, 2H),
5.11 (s, 2H), 4.95 (m, 1H), 4.05 (m, 3H), 3.58 (m, 4H), 3.42 (m, 4H),
2.28 (m, 2H), 1.96 (m, 1H), 1.81 (m, 1H), 1.37 (s, 9H), 1.19 (t, J =
7.0 Hz, 3H). tR (method j): 1.77 min. MS (ES) m/z: 678.3 (MH+).
6.1.6.2. 4-{(S)-4-tert-Butoxycarbonyl-2-[(5-carboxymethoxy-1phenyl-1H-pyrazole-3-carbonyl)-amino]-butyryl}-piperazine-1-carboxylic Acid Ethyl Ester. To a solution of 4-{(S)-2-[(5-benzyloxycarbonylmethoxy-1-phenyl-1H-pyrazole-3-carbonyl)-amino]-4-tertbutoxycarbonyl-butyryl}-piperazine-1-carboxylic acid ethyl ester (13.2
g, 19.5 mmol) in ethyl acetate (75 mL) was added under argon Pd/C
(1.1 g, 10%), and the suspension was stirred under an atmosphere of
hydrogen (3 bar) for 16 h. The suspension was ltered over a plug of
Celite and washed with ethyl acetate. The crude product obtained after
evaporation of the solvent was dried under vacuo at 40 C for 24 h to
give the title compound as a of a colorless solid (12.1 g, 100%). 1H
NMR (DMSO-d6): 8.14 (d, J = 8.2 Hz, 1H), 7.80 (d, J = 8.0 Hz,
2H), 7.53 (t, J = 8.0 Hz, 2H), 7.41 (t, J = 8.0 Hz, 1H), 6.34 (s, 1H),
4.94 (m, 1H), 4.90 (s, 2H), 4.05 (m, 3H), 3.60 (m, 4H), 3.39 (m, 4H),
2.29 (m, 2H), 1.94 (m, 1H), 1.81 (m, 1H), 1.38 (s, 9H), 1.19 (t, J =
7.0 Hz, 3H). tR (method j): 1.39 min. MS (ES) m/z: 588.3 (MH+).
6.1.6.3. 4-[(S)-4-Carboxy-2-({5-[2-((R)-3-hydroxy-pyrrolidin-1-yl)2-oxo-ethoxy]-1-phenyl-1H-pyrazole-3-carbonyl}-amino)-butyryl]piperazine-1-carboxylic Acid Ethyl Ester (17h). To a solution of 4{(S)-4-tert-butoxycarbonyl-2-[(5-carboxymethoxy-1-phenyl-1H-pyrazole-3-carbonyl)-amino]-butyryl}-piperazine-1-carboxylic acid ethyl
ester (90 mg, 0.15 mmol) in DMF (5 mL) were added DIPEA (54
L, 0.30 mmol), HOBt (23 mg, 0.15 mmol), and EDC (29 mg, 0.15
mmol). After 20 min, (R)-()-3-pyrrolidinol hydrochloride (19 mg,
0.15 mmol) was added, and the reaction mixture was stirred for 12 h.
After dilution with ethyl acetate, the reaction mixture was extracted
with aqueous LiCl (4% w/w) and aqueous NaHCO3. The organic
layer was dried over MgSO4, and the solvent was removed under
reduced pressure. The crude product was dissolved in dichloromethane (1.5 mL) and treated with triuoroacetic acid (127 L). After
they were stirred for 12 h, the solvents were removed under reduced
pressure, and the residue was puried by preparative reversed-phase
HPLC, eluting with a gradient of acetonitrile in water (+0.01%
triuoroacetic acid). After lyophilization, the product 17h (45 mg,
49%) was obtained as a white solid. 1H NMR (DMSO-d6): 8.13 (d, J
= 8.3 Hz, 1H), 7.87 (d, J = 8.3 Hz, 2H), 7.52 (t, J = 8.3 Hz, 2H), 7.39
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(t, J = 7.4 Hz, 1H), 6.35 (s, 1H, appears as 2 rotamers), 4.95 (m, 4H),
4.29 (s, 1H, appears as 2 rotamers), 4.05 (q, J = 6.9 Hz, 2H), 3.44 (m,
12H), 2.29 (m, 2H), 1.96 (m, 2H), 1.84 (m, 2H), 1.19 (t, J = 6.8 Hz,
3H). tR (method e): 1.22 min. MS (ES) m/z: 501.2 (MH+).
6.1.7. Representative Procedure: Preparation of 4-[(S)-4-Carboxy2-({5-[2-((S)-2-methylcarbamoyl-pyrrolidin-1-yl)-2-oxo-ethoxy]-1phenyl-1H-pyrazole-3-carbonyl}-amino)-butyryl]-piperazine-1-carboxylic Acid Ethyl Ester (18b) via Route 1 (Scheme 2). 6.1.7.1. 4-[(S)2-({5-[2-((S)-2-Benzyloxycarbonyl-pyrrolidin-1-yl)-2-oxo-ethoxy]-1phenyl-1H-pyrazole-3-carbonyl}-amino)-4-tert-butoxycarbonyl-butyryl]-piperazine-1-carboxylic Acid Ethyl Ester. To a solution of 4{(S)-4-tert-butoxycarbonyl-2-[(5-carboxymethoxy-1-phenyl-1H-pyrazole-3-carbonyl)-amino]-butyryl}-piperazine-1-carboxylic acid ethyl
ester (9.5 g, 16.2 mmol) in DMF (80 mL) were added HOBt (2.5
g, 16.2 mmol), DIPEA (5.7 mL, 32.4 mmol), and L-proline benzyl
ester hydrochloride (3.9 g, 16.2 mmol) at 0 C. At this temperature
was added EDC (3.1 g, 16.2 mmol) portionwise, and the suspension
was allowed to warm to room temperature over a period of 12 h. The
solvent was evaporated, and the residue was dissolved in ethyl acetate
and subsequently extracted with aqueous LiCl (4% w/w), aqueous
HCl (0.1 M), and aqueous NaHCO3. The organic layer was dried over
MgSO4, and the solvent was removed under reduced pressure to give
the crude product as amorphous solid (11.6 g, 92%). An analytically
pure sample could be obtained by purication of an aliquot by
preparative HPLC. 1H NMR (DMSO-d6): 8.13 (d, J = 7.8 Hz, 1H),
7.84 (d, J = 7.3 Hz, 2H), 7.52 (t, J = 7.3 Hz, 2H), 7.40 (t, J = 7.3 Hz,
1H), 7.33 (m, 5H), 6.38 (s, appears as rotamers, 1H), 5.11 (m, 4H),
4.95 (m, 1H), 4.42 (dd, J = 9.0 Hz, J = 4.3 Hz, 1H), 4.05 (q, J = 7.3
Hz, 2H), 3.56 (m, 10H), 2.331.74 (several multiplets, 8H), 1.38 (s,
9H), 1.18 (t, J = 7.0 Hz, 3H). tR (method j): 1.71 min. MS (ES) m/z:
775.3 (MH+).
6.1.7.2. 4-[(S)-4-tert-Butoxycarbonyl-2-({5-[2-((S)-2-carboxy-pyrrolidin-1-yl)-2-oxo-ethoxy]-1-phenyl-1H-pyrazole-3-carbonyl}amino)-butyryl]-piperazine-1-carboxylic Acid Ethyl Ester. To a
solution of 4-[(S)-2-({5-[2-((S)-2-benzyloxycarbonyl-pyrrolidin-1-yl)2-oxo-ethoxy]-1-phenyl-1H-pyrazole-3-carbonyl}-amino)-4-tert-butoxycarbonyl-butyryl]-piperazine-1-carboxylic acid ethyl ester (11.6 g,
14.9 mmol) in ethyl acetate (75 mL) was added under argon Pd/C (1
g, 10%), and the suspension was stirred under an atmosphere of
hydrogen (3 bar) for 24 h. The suspension was ltered over a plug of
Celite and washed with ethyl acetate. The crude product was obtained
after evaporation of the solvent (9.9 g, 89%) and used without further
purication. 1H NMR (DMSO-d6): 12.56 (s br, 1H), 8.11 (d, J = 7.8
Hz, 1H), 7.85 (d, J = 7.3 Hz, 2H), 7.52 (t, J = 7.3 Hz, 2H), 7.39 (t, J =
7.3 Hz, 1H), 6.37 (s, appears as rotamers, 1H), 5.06 (s, 2H), 4.94 (m,
1H), 4.28 (dd, J = 9.0 Hz, J = 4.1 Hz, 1H), 4.05 (q, J = 7.1 Hz, 2H),
3.57 (m, 5H), 3.42 (m, 5H), 2.311.74 (several multiplets, 8H), 1.37
(s, 9H), 1.18 (t, J = 7.0 Hz, 3H). tR (method j): 1.40 min. MS (ES) m/
z: 685.3 (MH+).
6.1.7.3. 4-[(S)-4-Carboxy-2-({5-[2-((S)-2-methylcarbamoyl-pyrrolidin-1-yl)-2-oxo-ethoxy]-1-phenyl-1H-pyrazole-3-carbonyl}-amino)butyryl]-piperazine-1-carboxylic Acid Ethyl Ester (18b). To a
solution of 4-[(S)-4-tert-butoxycarbonyl-2-({5-[2-((S)-2-carboxy-pyrrolidin-1-yl)-2-oxo-ethoxy]-1-phenyl-1H-pyrazole-3-carbonyl}amino)-butyryl]-piperazine-1-carboxylic acid ethyl ester (133 mg, 0.19
mmol) in DMF (5 mL) were added DIPEA (80 L, 0.49 mmol),
HOBt (45 mg, 0.29 mmol), and EDC (56 mg, 0.29 mmol). After 20
min, methylamine hydrochloride (20 mg, 0.29 mmol) was added, and
the reaction mixture was stirred for 12 h. After dilution with ethyl
acetate, the reaction mixture was extracted with aqueous LiCl (4% w/
w) and aqueous NaHCO3. The organic layer was dried over MgSO4,
and the solvent was removed under reduced pressure. The crude
product was dissolved in dichloromethane (4 mL) and treated with
triuoroacetic acid (128 L). After this was stirred for 12 h, the
solvents were removed under reduced pressure, and the residue was
puried by preparative reversed-phase HPLC, eluting with a gradient
of acetonitrile in water (+0.01% triuoroacetic acid). After
lyophilization, the product 18b (46 mg, 37%) was obtained as a
white solid. 1H NMR (DMSO-d6): 8.13 (d, J = 8.4 Hz, 1H), 7.85 (d,
J = 8.0 Hz, 2H), 7.76 (m, 1H), 7.53 (t, J = 7.6 Hz, 2H), 7.40 (t, J = 7.4
Hz, 1H), 6.41 (s, 1H), 5.00 (m, 3H), 4.22 (dd, J = 8.5 Hz, J = 3.2 Hz,
1H), 4.06 (q, J = 6.9 Hz, 2H), 3.51 (m, 10H), 2.55 (d, J = 4.6 Hz, 3H),
2.29 (m, 2H), 1.99 (m, 2H), 1.89 (m, 2H), 1.81 (m, 2H), 1.19 (t, J =
6.8 Hz, 3H). tR (method e): 1.25 min. MS (ES) m/z: 642.3 (MH+).
6.1.8. Representative Procedure: Preparation of 4-[(S)-4-Carboxy2-({5-[2-((S)-2-cyclobutylcarbamoyl-pyrrolidin-1-yl)-2-oxo-ethoxy]1-phenyl-1H-pyrazole-3-carbonyl}-amino)-butyrylamino]-piperidine-1-carboxylic Acid Ethyl Ester (19a) via Route 2 (Scheme 2).
6.1.8.1. (S)-2-({5-[2-((S)-2-Cyclobutylcarbamoyl-pyrrolidin-1-yl)-2oxo-ethoxy]-1-phenyl-1H-pyrazole-3-carbonyl}-amino)-pentanedioic Acid 5-tert-Butyl Ester 1-Methyl Ester. To a solution of 5-[2((S)-2-cyclobutylcarbamoyl-pyrrolidin-1-yl)-2-oxo-ethoxy]-1-phenyl1H-pyrazole-3-carboxylic acid (1.1 g, 2.7 mmol) in DMF (20 mL)
were added DIPEA (1 mL, 6.1 mmol), HOBt (0.41 g, 2.7 mmol),
EDC (0.51 g, 2.7 mmol), and H-Glu(OtBu)-OMe hydrochloride (0.68
g, 2.7 mmol). After it was stirred for 24 h, the solution was
concentrated, taken up with dichloromethane, and subsequently
extracted with aqueous LiCl (4% w/w), aqueous HCl (0.1 M), and
saturated NaHCO3. The crude product was puried by ash
chromatography on silica using an ethyl acetate/heptane 50:50 to
100:0 gradient to give the title compound as a colorless foam (1.88 g,
100%). tR (method j): 1.55 min. MS (ES) m/z: 612.3 (MH+).
6.1.8.2. (S)-2-({5-[2-((S)-2-Cyclobutylcarbamoyl-pyrrolidin-1-yl)-2oxo-ethoxy]-1-phenyl- 1H-pyrazole-3-carbonyl}-amino)-pentanedioic Acid 5-tert-Butyl Ester. To a solution of (S)-2-({5-[2-((S)-2cyclobutylcarbamoyl-pyrrolidin-1-yl)-2-oxo-ethoxy]-1-phenyl-1H-pyrazole-3-carbonyl}-amino)-pentanedioic acid 5-tert-butyl ester 1-methyl
ester (1.84 g, 3.0 mmol) in THF (12 mL) was added LiOH (73 mg,
3.0 mmol) in water (4 mL). After 2 h, the reaction mixture was
neutralized with Amberlite IR-120, ltered, and washed with methanol.
After evaporation of the solvent, the title compound was obtained as a
colorless oil (1.80 g, 100%). 1H NMR (DMSO-d6): 8.24 (d, J = 7.5
Hz, 1H), 8.02 (d, J = 7.5 Hz, appears as rotamers, 1H), 7.91 (d, J = 8.3
Hz, 2H), 7.57 (t, J = 8.3 Hz, 2H), 7.43 (t, J = 8.3 Hz, 1H), 6.44 (s, 1H,
appears as rotamers), 5.09 (s, 2H), 4.44- 4.11 (m, 3H), 3.60 (m, 1H),
3.48 (m, 2H), 2.321.52 (several multiplets, 14H), 1.37 (s, 9H). tR
(method j): 1.38 min. MS (ES) m/z: 598.2 (MH+).
6.1.8.3. 4-[(S)-4-Carboxy-2-({5-[2-((S)-2-cyclobutylcarbamoyl-pyrrolidin-1-yl)-2-oxo-ethoxy]-1-phenyl-1H-pyrazole-3-carbonyl}amino)-butyrylamino]-piperidine-1-carboxylic Acid Ethyl Ester
(19a). To a solution of (S)-2-({5-[2-((S)-2-cyclobutylcarbamoylpyrrolidin-1-yl)-2-oxo-ethoxy]-1-phenyl-1H-pyrazole-3-carbonyl}amino)-pentanedioic acid 5-tert-butyl ester (150 mg, 0.25 mmol) in
DMF (7 mL) were added DIPEA (62 L, 1.1 mmol), HATU (95 mg,
0.36 mmol), and ethyl 4-amino-1-piperidinecarboxylate (43 mg, 0.25
mmol). After it was stirred for 12 h, the saturated NaHCO3 solution
was added, and the mixture loaded on a chem elut cartridge, the crude
product being eluted with dichloromethane. The solution was
concentrated to a volume of 1 mL and stirred in the presence of
triuoroacetic acid (190 L). After it was stirred for 4 h, the solvents
were removed under reduced pressure, and the residue was puried by
preparative HPLC (C18 reverse phase column, elution with a water/
acetonitrile gradient with 0.1% triuoroacetic acid). After lyophilization, the product 19a (35 mg, 20%) was obtained as a white solid. 1H
NMR (DMSO-d6): 8.09 (d, J = 8.0 Hz, 1H), 7.98 (d, J = 8.4 Hz,
1H), 7.85 (d, J = 8.4 Hz, 2H), 7.52 (t, J = 7.6 Hz, 2H), 7.38 (t, J = 7.6
Hz, 1H), 6.41 (s, 1H), 5.04 (s, 2H), 4.96 (m, 1H), 4.21 (dd, J = 8.4
Hz, J = 3.9 Hz, 1H), 4.14 (m, 1H), 4.05 (q, J = 6.9 Hz, 2H), 3.82 (m,
1H), 3.52 (m, 8H), 2.29 (m, 2H), 2.11 - 1.80 (m, 8H), 1.72 (m, 3H),
1.59 (m, 2H), 1.25 (m, 2H), 1.19 (t, J = 6.8 Hz, 3H). tR (method b):
1.59 min. MS (ES) m/z: 696.5 (MH+).
6.2. Biology: Human P2Y12 Recombinant Cell Membrane
Binding Assay.17 As a source of P2Y12, a membrane preparation was
prepared from CHO cells with recombinant expression of the human
P2Y12 receptor according to standard procedures. To a 96-well
microtiter plate, added were the following: (a) 24 L of assay buer
[10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
(HEPES), 138 mM NaCl, 2.9 mN KCl, 12 mM NaHCO3, 1 mM
EDTA-Na, and 0.1% BSA, pH 7.4], (b) 1 L of compound in DMSO,
(c) 50 L of P2Y12 CHO membrane (20 g/mL) and after 15 min at
room temperature, and (d) 25 L of 1.61 nM [33P]2-MeS-ADP
(Perkin-Elmer NEN custom synthesis, specic activity 2100 Ci/
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mmol) made in assay buer. The nal concentration of [33P]2-MeSADP was 0.8 nM, and the Kd value determined for [33P]2-MeS-ADP
was 0.5 nM. After 20 min of incubation at room temperature, samples
were transferred and aspirated to 96-well microtiter lterplates
(Millipore HTS GF/B) and prewetted for 20 min with 300 L of
stop buer (10 mM HEPES, 138 mM NaCl, pH 7.4). After they were
washed four times with 400 L/well of stop buer, the lters were airdried overnight. After the addition of 0.1 mL of Microscint 20
Scintillation Fluid (Perkin-Elmer #6013621), the lter plates were
incubated for 2 h at room temperature and counted in a Microbeta
Scintillation Counter. The binding of compound is expressed as a %
inhibition of specic binding, dened by subtraction of the background
with 1 mM ADP. Ki values were calculated from the IC50 values using
the ChengPruso equation assuming binding to a single binding site
and were averaged from at least triplicate determination. The mean Ki
value of the positive control used for each run was 0.4 M, and the
standard error of the mean (SEM) for this standard compound was
0.016 M (n = 61).
6.2.1. Inhibition of Human Platelet Aggregation. 6.2.1.1. hPRP
Preparation. Human whole blood was from the Sano-Aventis inhouse blood donor service. The protocol was approved by the local
ethics committee, and all blood donors signed informed consent.
Whole blood was collected from healthy volunteers using 20 mL
syringes containing 2 mL of acidcitratedextrose solution (ACD-A)
(for 96-well assays) or 2 mL of buered citrate (for the light
transmission aggregometry, Born method). The anticoagulated whole
blood was transferred into 15 mL polypropylene conical tubes (10 mL
per tube). The tubes were centrifuged for 15 min at room temperature
at 150g (for 96-well assays) or at 340g (for the Born method), leading
to a supernatant of hPRP. The hPRP layer was collected from each
tube and pooled for each donor. The platelet concentration was
determined using a Coulter counter. The 15 mL tubes containing the
pellet of cellular components were centrifuged again for 10 min at
1940g, leaving a supernatant of platelet poor plasma (PPP). The PPP
was collected for each donor. The PRP was diluted with PPP to a nal
concentration of 3 108 platelets/mL with the PPP.
6.2.1.2. IPs Preparation. IPs were obtained by centrifugation of
PRP at 430g for 20 min at room temperature. The resulting platelet
pellet was dissolved in a modied Tyrode's buer containing 145 mM
NaCl, 5 mM KCl, 0.1 mM MgCl6H2O, 5.5 mM glucose, and 15 mM
Hepes. The buer was adjusted to pH 7.4. Platelets were counted and
adjusted to 300 103 platelets/L. To 320 L of IPs, 20 L of CaCl2
(nal concentration, 0.5 mM) was added and incubated at 37 C for 2
min before adding 20 L of brinogen (nal concentration, 1 mg/
mL). The respective P2Y12 antagonist was added (20 L) and
incubated, and the response to platelet activating agonists (20 L) was
recorded.
6.2.1.3. Human Platelet Aggregation 96-Well Assay.10 The
human platelet aggregation assay was performed in 96-well plates
using a microtiter plate reader (SpectraMax Plus 384 with SoftMax Pro
software from Molecular Devices). In the plate, 15 L of test
compound at 10 nal concentration in NaCl was mixed with 120 L
of fresh hPRP and incubated for 5 min. Following that incubation
period, 15 L of 40 M ADP was added to the reaction mix, leading to
a nal concentration of 4 M ADP. The plates were then transferred
to the microplate reader, and aggregation was measured over 20 min.
The instrument settings include the following: absorbance at 650 nm,
run time 20 min with readings in 1 min intervals, and 50 s of shaking
between readings, all performed at 37 C. Results of the assay are
expressed as % inhibition and are calculated using the AUC of the
absorbance over 20 min. The IC50 values were averaged from at least
triplicate determination. The mean value of the positive control used
for each run was 0.27 M, and the SEM for this standard compound
was 0.026 M (n = 96).
6.2.1.4. Human Platelet Aggregation Assay Light Transmission
Aggregometry (Born Method).10 The human platelet aggregation
assay was performed in single use cuvettes using the platelet
aggregation proler (PAP-8, Bio/Data corporation, Horsham, PA).
In the assay cuvette, 4 L of the test compound at 100 nal
concentration in DMSO was mixed with 392 L of fresh hPRP and
incubated for 2 min at 37 C with 1.200 rpm stirring. Following that

incubation period, 4 L of a 250 M ADP solution was added to the
reaction mix, leading to a nal concentration of 2.5 M ADP. After
that, aggregation was measured over 6 min at 37 C with 1.200 rpm
stirring. Results of the assay are expressed as % inhibition and are
calculated using the AUC of the absorbance over 6 min. The IC50
values were averaged from at least triplicate determination. The
determination of the activity on ADP-, Col-, and Thr-induced
aggregation in IPs was performed analogously. The nal concentration
of ADP was 10 M, of Col was 1 g/mL, and of Thr was 0.1 U/mL.
6.3. Computational Procedures: Homology Modeling and
Molecular Docking. A homology model of the P2Y12 receptor was
generated using the crystal structure of the CXCR4 (PDB: 1odu) as
the structural template. Sequence alignment and 3D model generation
were performed using the software Prime (Prime, v2.2, Schrodinger,
LLC, New York, NY). Molecular docking was performed with the
Induced Fit Docking procedure22 based on Glide v5.7 (Glide, v5.7,
Schrodinger, LLC) and Prime v2.2, as implemented in the Schrodinger
package.
6.3.1. Generation of 3D Ligand Alignments. For the generation of
multiple ligand 3D superimpositions, we used the MARS approach,21
which is based on the pairwise alignment of all molecules within the
data set using the software tool ROCS.23
6.3.2. Generation of 3D QSAR Models. Default settings were used
for CoMFA and CoMSIA in Sybyl24 if not otherwise indicated. Steric
and electrostatic energies in CoMFA were calculated at grid points
with 2 spacing, a positively charged carbon atom, and a distancedependent dielectric constant with MMFF94 charges. For CoMSIA,
steric, electrostatic, hydrophobic, donor, and acceptor elds were built
using a Gaussian distance-dependent function. Cross-validated
analyses were run using SAMPLS (LOO) or 10 cross-validation
groups (LNO) with random selection of group members.
ASSOCIATED CONTENT
S Supporting Information
*
Experimental data for selected compounds 1720 and

additional biological characterization data. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: Marc.Nazare@sano.com.
Notes
The authors declare no competing nancial interest.
ACKNOWLEDGMENTS
We thank A. Sihorsch, M. Kammerer-Dienst, C. Prosser, S.
Herok, D. Thorn, H. Krause, G. Sibenhorn, S. Stamm, A.
Haber, F. Hopnger, M. Pfeier, and R. Katzenmeier for their
excellent technical assistance.
ABBREVIATIONS USED
ACD-A, acidcitratedextrose solution; ADP, adenosine
diphosphate; AUC, area under curve; CHO, Chinese hamster
ovary cell; Col, collagen; CoMFA, comparative molecular eld
analysis; CoMSIA, comparative molecular similarity index
analysis; CXCR4, C-X-C chemokine receptor type 4; EC,
extracellular loop; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; HATU, O-(7-azabenzotriazol-1yl)-N,N,N,N-tetramethyluronium hexauorophosphate;
HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid;
HOBt, 1-hydroxybenzotriazole; IP, isolated platelets; MARS,
multiple alignments by ROCS-based similarity; ND, not
determined; P2Y1, P2Y purinoceptor 1; P2Y12, P2Y purinoceptor 12; PI3K, phosphatidylinositol 3-kinase; hPRP, human
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K. A.; Hiebsch, R. R.; Huff, R. M.; Lachance, R. M.; Mischke, D. A.;
Rapp, S. R.; Woerndle, R. S.; Ennis, M. D. Piperazinyl-glutamatepyridines as potent orally bioavailable P2Y12 antagonists for inhibition
of platelet aggregation. Bioorg. Med. Chem. Lett. 2009, 19, 46574663.
(b) Parlow, J. J.; Burney, M. W.; Case, B. L.; Girard, T. J.; Hall, K. A.;
Harris, P. K.; Hiebsch, R. R.; Huff, R. M.; Lachance, R. M.; Mischke,
D. A.; Rapp, S. R.; Woerndle, R. S.; Ennis, M. D. Part II: Piperazinylglutamate-pyridines as potent orally bioavailable P2Y12 antagonists for
inhibition of platelet aggregation. Bioorg. Med. Chem. Lett. 2010, 20,
13881394. (c) Parlow, J. J.; Burney, M. W.; Case, B. L.; Girard, T. J.;
Hall, K. A.; Harris, P. K.; Hiebsch, R. R.; Huff, R. M.; Lachance, R. M.;
Mischke, D. A.; Rapp, S. R.; Woerndle, R. S.; Ennis, M. D. Piperazinyl
glutamate pyridines as potent orally bioavailable P2Y12 antagonists for
inhibition of platelet aggregation. J. Med. Chem. 2010, 53, 20102037.
(12) Part of this work has been patented by the authors: (a) Nazare,
M.; Zech, G.; Goerlitzer, J.; Just, M.; Weiss, T.; Hessler, G.;
Czechtizky, W.; Ruf, S. Pyrazole-carboxamide derivatives as P2Y12
antagonist. WO2009080227, 2009. (b) Nazare, M.; Zech, G.; Just, M.;
platelet-rich plasma; Thr, thrombin; TM, transmembrane

domain; TOTU, O-((ethoxycarbonyl)cyanomethyleneamino)N,N,N,N-tetramethyluronium tetrauoroborate; Z, Cbz benzyloxy-carbonyl
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Article

Identication of High-Anity P2Y12 Antagonists Based on a

ABSTRACT: A series of novel, highly potent P2Y12 antagonists as

The widespread successful clinical use of the irreversible

The activation and aggregation of blood platelets are essential

Received: June 4, 2012

dx.doi.org/10.1021/jm300771j | J. Med. Chem. 2012, 55, 86158629

Journal of Medicinal Chemistry

In seeking suitable P2Y12 drug candidates in this area, we12

receptor type 4 (CXCR4) receptor as a structural template (see

2. RESULTS AND DISCUSSION

dx.doi.org/10.1021/jm300771j | J. Med. Chem. 2012, 55, 86158629

Journal of Medicinal Chemistry

Scheme 1. Synthetic Routes to Key Building Blocksa

points toward the extracellular region. Interestingly, a similar

Reagents and conditions: (a) TOTU, N-ethylmorpholine, DMF,

dx.doi.org/10.1021/jm300771j | J. Med. Chem. 2012, 55, 86158629

Journal of Medicinal Chemistry

Scheme 2. Synthetic Routes Used for the Assembly of the

Table 1. Binding and hPRP Activity of Explorative

synthesize a wide range of P2Y12 antagonists and to obtain a

3. RESULTS AND DISCUSSION

contrast, the simple primary amide 17f or the acyclic tertiary

dx.doi.org/10.1021/jm300771j | J. Med. Chem. 2012, 55, 86158629

Journal of Medicinal Chemistry

we turned our attention to proline derivatives, which appeared

the initial pyrrolidine derivatives 17h and 17i. However, a

dx.doi.org/10.1021/jm300771j | J. Med. Chem. 2012, 55, 86158629

Journal of Medicinal Chemistry

amide 18b over a linear ethyl amide 18c to a n-propyl amide

extension of the ring size from a cyclopropyl amide (18g) to a

dx.doi.org/10.1021/jm300771j | J. Med. Chem. 2012, 55, 86158629

Journal of Medicinal Chemistry

corresponding 2- or 3-methyl-substituted piperazines 19mp

dx.doi.org/10.1021/jm300771j | J. Med. Chem. 2012, 55, 86158629

Journal of Medicinal Chemistry

others,11a,c the corresponding glycine analogues were found to

Table 4. Binding and hPRP Activity of Compounds with

Table 5. Activity and Selectivity Data of Selected

the contribution of a reduced association of the compounds to

dx.doi.org/10.1021/jm300771j | J. Med. Chem. 2012, 55, 86158629

Journal of Medicinal Chemistry

binding appears to contribute to the observed high antagonistic

4. DEVELOPMENT OF 3D QSAR MODELS

Figure 3. (a) Shows steric and electrostatic std*coe contour maps

residues attached to the 5-O-position of the pyrazolone like the

dx.doi.org/10.1021/jm300771j | J. Med. Chem. 2012, 55, 86158629

Journal of Medicinal Chemistry

dx.doi.org/10.1021/jm300771j | J. Med. Chem. 2012, 55, 86158629

Journal of Medicinal Chemistry

nyl-1H-pyrazole-3-carboxylic acid ethyl ester 84.5 g, 11.6 mmol) in

acetate gradient to give the corresponding O-alkylation product (860

dx.doi.org/10.1021/jm300771j | J. Med. Chem. 2012, 55, 86158629

Journal of Medicinal Chemistry

dx.doi.org/10.1021/jm300771j | J. Med. Chem. 2012, 55, 86158629

Journal of Medicinal Chemistry

incubated for 2 min at 37 C with 1.200 rpm stirring. Following that

Experimental data for selected compounds 1720 and

The authors declare no competing nancial interest.

dx.doi.org/10.1021/jm300771j | J. Med. Chem. 2012, 55, 86158629

Journal of Medicinal Chemistry

platelet-rich plasma; Thr, thrombin; TM, transmembrane

(1) Gachet, C. ADP receptors of platelets and their inhibition.